Motor proteins and methods for their use

ABSTRACT

The invention provides isolated nucleic acid and amino acid sequences of HsKif12a, antibodies to HsKif12a, methods of screening for HsKif12a modulators using biologically active HsKif12a, and kits for screening for HsKif12a modulators.

FIELD OF THE INVENTION

The invention provides isolated nucleic acid and amino acid sequences ofHsKif12a, methods of detecting HsKif12a and screening for HsKif12amodulators using biologically active HsKif12a, and kits for screeningfor HsKif12a modulators.

BACKGROUND OF THE INVENTION

The kinesin superfamily is an extended family of related microtubulemotor proteins. It can be classified into at least 8 subfamilies basedon primary amino acid sequence, domain structure, velocity of movement,and cellular function. This family is exemplified by “true” kinesin,which was first isolated from the axoplasm of squid, where it isbelieved to play a role in anterograde axonal transport of vesicles andorganelles (see, e.g., Goldstein, Annu. Rev. Genet. 27:319-351(1993)).Kinesin uses ATP to generate force and directional movement associatedwith microtubules.

Within this functional group of kinesins resides a group of kinesinsfrom several organisms that share significant sequence homology,including mouse kinesin Kif12. See, Nakagawa et al. (1997) Proc. Natl.Acad. Sci. USA 94:9654-9659 and GenBank Accession Number AB001428, eachof which is incorporated herein by reference for all purposes.

The discovery of a new kinesin motor protein which is the human orthologof mouse kinesin Kif12, and the polynucleotides encoding it satisfies aneed in the art by providing new compositions which are useful in thediagnosis, prevention, and treatment of cancer, neurological disorders,and disorders of vesicular transport.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of a new human kinesinmotor protein, HsKif12a, the polynucleotide encoding HsKif12a, and theuse of these compositions for the diagnosis, treatment, or prevention ofcancer, neurological disorders, and disorders of vesicular transport.

In one aspect, the invention provides an isolated nucleic acid sequenceencoding a kinesin superfamily motor protein, wherein the motor proteinhas the following properties: (i) the protein's activity includesmicrotubule stimulated ATPase activity; and (ii) the protein has asequence that has greater than 70%, 80%, or 90% amino acid sequenceidentity to SEQ ID NO:2 as measured using a sequence comparisonalgorithm. In one embodiment, the protein further specifically binds topolyclonal antibodies raised against SEQ ID NO:2

In one embodiment, the nucleic acid encodes HsKif12a or a fragmentthereof. In another embodiment, the nucleic acid encodes SEQ ID NO:2. Inanother embodiment, the nucleic acid has a nucleotide sequence of SEQ IDNO:1.

In one aspect, the nucleic acid comprises a sequence which encodes anamino acid sequence which has greater than 70% sequence identity withSEQ ID NO:2, preferably greater than 80%, more preferably greater than90%, more preferably greater than 95% or, in another embodiment, has 98to 100% sequence identity with SEQ ID NO:2.

In one embodiment, the nucleic acid comprises a sequence which hasgreater than 55 or 60% sequence identity with SEQ ID NO: 1, preferablygreater than 70%, more preferably greater than 80%, more preferablygreater than 90 or 95% or, in another embodiment, has 98 to 100%sequence identity with SEQ ID NO: 1. In another embodiment providedherein, the nucleic acid hybridizes under stringent conditions to anucleic acid having a sequence or complementary sequence of SEQ ID NO:1.

In another aspect, the invention provides an expression vectorcomprising a nucleic acid encoding a kinesin superfamily motor protein,wherein the motor protein has the following properties: (i) theprotein's activity includes microtubule stimulated ATPase activity; and(ii) the protein has a sequence that has greater than 70, 80, or 90%amino acid sequence identity to SEQ ID NO:2 as measured using a sequencecomparison algorithm. The invention further provides a host celltransfected with the vector.

In another aspect, the invention provides an isolated kinesinsuperfamily motor protein, wherein the protein has one or more of theproperties described above. In one embodiment, the protein specificallybinds to polyclonal antibodies generated against a motor domain, taildomain or other fragment of HsKif12a. In another embodiment, the proteincomprises an amino acid sequence of SEQ ID NO:2.

In one aspect, the protein provided herein comprises an amino acidsequence which has greater than 70% sequence identity with SEQ ID NO:2,preferably greater than 80%, more preferably greater than 90%, morepreferably greater than 95% or, in another embodiment, has 98 to 100%sequence identity with SEQ ID NO:2.

The invention features a substantially purified polypeptide comprisingthe amino acid sequence of SEQ ID NO:2 or a fragment thereof and moreparticularly, the motor domain of the amino acid sequence of SEQ ID NO:2or a fragment thereof.

In one embodiment, the present invention provides a method ofidentifying a candidate agent as a modulator of the activity of a targetprotein. The method comprises adding a candidate agent to a mixturecomprising a target protein which directly or indirectly produces ADP orphosphate, under conditions that normally allow the production of ADP orphosphate. The method further comprises subjecting the mixture to areaction that uses said ADP or phosphate as a substrate under conditionsthat normally allow the ADP or phosphate to be utilized and determiningthe level of activity of the reaction as a measure of the concentrationof ADP or phosphate. A change in the level between the presence andabsence of the candidate agent indicates a modulator of the targetprotein.

The phrase “use ADP or phosphate” means that the ADP or phosphate aredirectly acted upon by detection reagents. In one case, the ADP, forexample, can be hydrolyzed or can be phosphorylated. As another example,the phosphate can be added to another compound. As used herein, in eachof these cases, ADP or phosphate is acting as a substrate.

Preferably, the target protein either directly or indirectly producesADP or phosphate and comprises a motor domain. More preferably, thetarget protein comprises a kinesin superfamily motor protein asdescribed above and most preferably, the target protein comprisesHsKif12a or a fragment thereof.

Also provided are modulators of the target protein including agents forthe treatment of cellular proliferation, including cancer, hyperplasias,restenosis, cardiac hypertrophy, immune disorders and inflammation. Theagents and compositions provided herein can be used in variety ofapplications which include the formulation of sprays, powders, and othercompositions. Also provided herein are methods of treating cellularproliferation disorders such as cancer, hyperplasias, restenosis,cardiac hypertrophy, immune disorders and inflammation, for treatingdisorders associated with HsKif12a activity, and for inhibitingHsKif12a.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an embodiment of a nucleic acid sequence encoding aparticularly preferred fragment of the motor domain of HsKif12a (SEQ IDNO:1).

FIG. 2 shows the amino acid sequence of a particularly preferredfragment of the motor domain of HsKif12a (SEQ ID NO:2).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“ADP” refers to adenosine diphosphate and also includes ADP analogs,including, but not limited to, deoxyadenosine diphosphate (dADP) andadenosine analogs. “Antibody” refers to a polypeptide substantiallyencoded by an immunoglobulin gene or immunoglobulin genes, or fragmentsthereof which specifically bind and recognize an analyte (antigen). Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively. The term antibody also includesantibody fragments either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAmethodologies.

An “anti-HsKif12a” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by the HsKif12a gene, cDNA, ora subsequence thereof.

“Biologically active” target protein refers to a target protein that hasone or more of kinesin protein's biological activities, including, butnot limited to microtubule stimulated ATPase activity, as tested, e.g.,in an ATPase assay. Biological activity can also be demonstrated in amicrotubule gliding assay or a microtubule binding assay. “ATPaseactivity” refers to ability to hydrolyze ATP. Other activities includepolymerization/depolymerization (effects on microtubule dynamics),binding to other proteins of the spindle, binding to proteins involvedin cell-cycle control, or serving as a substrate to other enzymes, suchas kinases or proteases and specific kinesin cellular activities, suchas chromosome congregation, axonal transport, etc.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains a target protein or a fragment thereof or nucleicacid encoding a target protein or a fragment thereof. Biological samplesmay also include sections of tissues such as frozen sections taken forhistological purposes. A biological sample comprises at least one cell,preferably plant or vertebrate. Embodiments include cells obtained froma eukaryotic organism, preferably eukaryotes such as fungi, plants,insects, protozoa, birds, fish, reptiles, and preferably a mammal suchas rat, mice, cow, dog, guinea pig, or rabbit, and most preferably aprimate such as chimpanzees or humans.

A “comparison window” includes reference to a segment of any one of thenumber of contiguous positions selected from the group consisting offrom 25 to 600, usually about 50 to about 200, more usually about 100 toabout 150 in which a sequence may be compared to a reference sequence ofthe same number of contiguous positions after the two sequences areoptimally aligned. Methods of alignment of sequences for comparison arewell-known in the art. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity methods of Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444 (1988) and Altschul et al. Nucleic Acids Res. 25(17): 3389-3402(1997), by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and BLAST in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Ausubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a dendrogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360 (1987). The method used is similar to themethod described by Higgins & Sharp, CABIOS 5:151-153 (1989). As ageneral rule, PileUp can align up to 500 sequences, with any singlesequence in the final alignment restricted to a maximum length of 7,000characters.

The multiple alignment procedure begins with the pairwise alignment ofthe two most similar sequences, producing a cluster of two alignedsequences. This cluster can then be aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences canbe aligned by a simple extension of the pairwise alignment of twoindividual sequences. A series of such pairwise alignments that includesincreasingly dissimilar sequences and clusters of sequences at eachiteration produces the final alignment.

“Variant” applies to both amino acid and nucleic acid sequences. Withrespect to particular nucleic acid sequences, conservatively modifiedvariants refers to those nucleic acids which encode identical oressentially identical amino acid sequences, or where the nucleic aciddoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given protein. Forinstance, the codons GCA, GCC, GCG and GCT all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide. Such nucleic acidvariations are “silent variations,” which are one species ofconservatively modified variations. Every nucleic acid sequence hereinwhich encodes a polypeptide also describes every possible silentvariation of the nucleic acid. One of skill will recognize that eachdegenerate codon in a nucleic acid can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

Also included within the definition of target proteins of the presentinvention are amino acid sequence variants of wild-type target proteins.These variants fall into one or more of three classes: substitutional,insertional or deletional variants. These variants ordinarily areprepared by site specific mutagenesis of nucleotides in the DNA encodingthe target protein, using cassette or PCR mutagenesis or othertechniques well known in the art, to produce DNA encoding the variant,and thereafter expressing the DNA in recombinant cell culture. Varianttarget protein fragments having up to about 100-150 amino acid residuesmay be prepared by in vitro synthesis using established techniques.Amino acid sequence variants are characterized by the predeterminednature of the variation, a feature that sets them apart from naturallyoccurring allelic or interspecies variation of the target protein aminoacid sequence. The variants typically exhibit the same qualitativebiological activity as the naturally occurring analogue, althoughvariants can also be selected which have modified characteristics.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to about 20 amino acids,although considerably longer insertions may be tolerated. Deletionsrange from about 1 to about 20 residues, although in some cases,deletions may be much longer.

Substitutions, deletions, and insertions or any combinations thereof maybe used to arrive at a final derivative. Generally, these changes aredone on a few amino acids to minimize the alteration of the molecule.However, larger characteristics may be tolerated in certaincircumstances.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

(see, e.g., Creighton, Proteins (1984)).

“Cytoskeletal component” denotes any molecule that is found inassociation with the cellular cytoskeleton, that plays a role inmaintaining or regulating the structural integrity of the cytoskeleton,or that mediates or regulates motile events mediated by thecytoskeleton. Includes cytoskeletal polymers (e.g., actin filaments,microtubules, intermediate filaments, myosin fragments), molecularmotors (e.g., kinesins, myosins, dyneins), cytoskeleton associatedregulatory proteins (e.g., tropomysin, alpha-actinin) and cytoskeletalassociated binding proteins (e.g., microtubules associated proteins,actin binding proteins).

“Cytoskeletal function” refers to biological roles of the cytoskeleton,including but not limited to the providing of structural organization(e.g., microvilli, mitotic spindle) and the mediation of motile eventswithin the cell (e.g., muscle contraction, mitotic chromosome movements,contractile ring formation and function, pseudopodal movement, activecell surface deformations, vesicle formation and translocation.)

A “diagnostic” as used herein is a compound, method, system, or devicethat assists in the identification and characterization of a health ordisease state. The diagnostic can be used in standard assays as is knownin the art.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

“High stringency conditions” may be identified by those that: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C.

“High throughput screening” as used herein refers to an assay whichprovides for multiple candidate agents or samples to be screenedsimultaneously. As further described below, examples of such assays mayinclude the use of microtiter plates which are especially convenientbecause a large number of assays can be carried out simultaneously,using small amounts of reagents and samples.

By “host cell” is meant a cell that contains an expression vector andsupports the replication or expression of the expression vector. Hostcells may be prokaryotic cells such as E. coli, or eukaryotic cells suchas yeast, insect, amphibian, or mammalian cells such as CHO, HeLa andthe like, or plant cells. Both primary cells and cultured cell lines areincluded in this definition.

The phrase “hybridizing specifically to” refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex mixture (e.g., total cellular) DNA or RNA. Stringent conditionsare sequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength, pH, and nucleic acid concentration) at which 50%of the probes complementary to the target sequence hybridize to thetarget sequence at equilibrium. Typically, stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.05 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (e.g., 10 to 50 nucleotides) and at least about 60° C. forlong probes (e.g., greater than 50 nucleotides). Stringent conditionsmay also be achieved with the addition of destabilizing agents such asformamide.

The terms “identical” or percent “identity”, in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence over a comparisonwindow, as measured using one of the following sequence comparisonalgorithms or by manual alignment and visual inspection. Preferably, thepercent identity exists over a region of the sequence that is at leastabout 25 amino acids in length, more preferably over a region that is 50or 100 amino acids in length. This definition also refers to thecomplement of a test sequence, provided that the test sequence has adesignated or substantial identity to a reference sequence. Preferably,the percent identity exists over a region of the sequence that is atleast about 25 nucleotides in length, more preferably over a region thatis 50 or 100 nucleotides in length.

When percentage of sequence identity is used in reference to proteins orpeptides, it is recognized that residue positions that are not identicaloften differ by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g,. charge or hydrophobicity) and therefore donot change the functional properties of the molecule. Where sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well known to thoseof skill in the art. The scoring of conservative substitutions can becalculated according to, e.g., the algorithm of Meyers & Millers,Computer Applic. Biol. Sci. 4:11-17 (1988), e.g., as implemented in theprogram PC/GENE (Intelligenetics, Mountain View, Calif.).

The terms “isolated”, “purified”, or “biologically pure” refer tomaterial that is substantially or essentially free from components whichnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified. In anisolated gene, the nucleic acid of interest is separated from openreading frames which flank the gene of interest and encode proteinsother than the protein of interest. The term “purified” denotes that anucleic acid or protein gives rise to essentially one band in anelectrophoretic gel. Particularly, it means that the nucleic acid orprotein is at least 85% pure, more preferably at least 95% pure, andmost preferably at least 99% pure.

A “label” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example, usefullabels include fluorescent proteins such as green, yellow, red or bluefluorescent proteins, radioisotopes such as ³²p, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or haptens and proteins for which antisera ormonoclonal antibodies are available (e.g., the polypeptide of SEQ IDNO:2 can be made detectable, e.g., by incorporating a radio-label intothe peptide, and used to detect antibodies specifically reactive withthe peptide).

A “labeled nucleic acid probe or oligonucleotide” is one that is bound,either covalently, through a linker, or through ionic, van der Waals, orhydrogen bonds to a label such that the presence of the probe may bedetected by detecting the presence of the label bound to the probe.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and %SDS)less stringent than those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 μg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

“Modulators,” “inhibitors,” and “activators of a target protein” referto modulatory molecules identified using in vitro and in vivo assays fortarget protein activity. Such assays include ATPase activity,microtubule gliding, microtubule depolymerizing activity, and bindingactivity such as microtubule binding activity or binding of nucleotideanalogs. Samples or assays that are treated with a candidate agent at atest and control concentration. The control concentration can be zero.If there is a change in target protein activity between the twoconcentrations, this change indicates the identification of a modulator.A change in activity, which can be an increase or decrease, ispreferably a change of at least 20% to 50%, more preferably by at least50% to 75%, more preferably at least 75% to 100%, and more preferably150% to 200%, and most preferably is a change of at least 2 to 10 foldcompared to a control. Additionally, a change can be indicated by achange in binding specificity or substrate.

“Molecular motor” refers to a molecule that utilizes chemical energy togenerate mechanical force. According to one embodiment, the molecularmotor drives the motile properties of the cytoskeleton.

The phrase “motor domain” refers to the domain of a target protein thatconfers membership in the kinesin superfamily of motor proteins througha sequence identity of approximately 35-45% identity to the motor domainof true kinesin.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences as well asthe sequence explicitly indicated. For example, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260)2605-2608(1985); Cassol et al. 1992; Rossolini et al. Mol. Cell. Probes 8:91-98(1994)). The term nucleic acid is used interchangeably with gene, cDNA,and mRNA encoded by a gene.

“Nucleic acid probe or oligonucleotide” is defined as a nucleic acidcapable of binding to a target nucleic acid of complementary sequencethrough one or more types of chemical bonds, usually throughcomplementary base pairing, usually through hydrogen bond formation. Asused herein, a probe may include natural (i.e., A, G, C, or T) ormodified bases. In addition, the bases in a probe may be joined by alinkage other than a phosphodiester bond, so long as it does notinterfere with hybridization. Thus, for example, probes may be peptidenucleic acids in which the constituent bases are joined by peptide bondsrather than phosphodiester linkages. It will be understood by one ofskill in the art that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are preferably directly labeledwith isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidine complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. A target protein comprises a polypeptide demonstrated to haveat least microtubule stimulated ATPase activity. Amino acids may bereferred to herein by either their commonly known three letter symbolsor by Nomenclature Commission. Nucleotides, likewise, may be referred toby their commonly accepted single-letter codes, i.e., the one-lettersymbols recommended by the IUPAC-IUB.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA box element. A promoter also optionally includes distal enhancer orrepressor elements which can be located as much as several thousand basepairs from the start site of transcription. A “constitutive” promoter isa promoter that is active under most environmental and developmentalconditions. An “inducible” promoter is a promoter that is underenvironmental or developmental regulation. The term “operably linked”refers to a functional linkage between a nucleic acid expression controlsequence (such as a promoter, or array of transcription factor bindingsites) and a second nucleic acid sequence, wherein the expressioncontrol sequence directs transcription of the nucleic acid correspondingto the second sequence.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, antibodies raised toHsKif12a with the amino acid sequence encoded in SEQ ID NO:2 can beselected to obtain only those antibodies that are specificallyimmunoreactive with HsKif12a and not with other proteins, except forpolymorphic variants, orthologs, alleles, and closely related homologuesof HsKif12a. This selection may be achieved by subtracting outantibodies that cross react with molecules, for example, such as C.elegans unc-104 and human Kif1A. A variety of immunoassay formats may beused to select antibodies specifically immunoreactive with a particularprotein. For example, solid-phase ELISA immunoassays are routinely usedto select antibodies specifically immunoreactive with a protein (see,e.g., Harlow & Lane, Antibodies, A Laboratory manual (1988), for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity). Typically a specific or selectivereaction will be at least twice background signal or noise and moretypically more than 10 to 100 times background.

The phrase “selectively associates with” refers to the ability of anucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

“Test composition” (used interchangeably herein with “candidate agent”and “test compound” and “test agent”) refers to a molecule orcomposition whose effect on the interaction between one or morecytoskeletal components it is desired to assay. The “test composition”can be any molecule or mixture of molecules, optionally in a carrier.

A “therapeutic” as used herein refers to a compound which is believed tobe capable of modulating the cytoskeletal system in vivo which can haveapplication in both human and animal disease. Modulation of thecytoskeletal system would be desirable in a number of conditionsincluding, but not limited to: abnormal stimulation of endothelial cells(e.g., atherosclerosis), solid and hematopoetic tumors and tumormetastasis, benign tumors, for example, hemangiomas, acoustic neuromas,neurofibromas, pyogenic granulomas, vascular malfunctions, abnormalwound healing, inflammatory and immune disorders such as rheumatoidarthritis, Bechet's disease, gout or gouty arthritis, abnormalangiogenesis accompanying: rheumatoid arthritis, psoriasis, diabeticretinopathy, and other ocular angiogenic disease such as, maculardegeneration, corneal graft rejection, corneal overgrowth, glaucoma, andOsler Webber syndrome.

II. The Target Protein

The present invention provides for the first time a nucleic acidencoding HsKif12a. This protein is a member of the kinesin superfamilyof motor proteins. More specifically, the HsKif12a sequence of FIG. 2shares approximately 89% identity to the mouse Kif12. See Nakagawa etal. (1997) Proc. Natl. Acad. Sci. USA 94:9654-9.

In one aspect, HsKif12a can be defined by having at least one orpreferably more than one of the following functional and structuralcharacteristics. Functionally, HsKif12a will have microtubule-stimulatedATPase activity, and microtubule motor activity that is ATP dependent.HsKif12a activity can also be described in terms of its ability to bindmicrotubules.

The novel nucleotides sequences provided herein encode HsKif12a orfragments thereof. Thus, in one aspect, the nucleic acids providedherein are defined by the novel proteins provided herein. The proteinprovided herein comprises an amino acid sequence which has one or moreof the following characteristics: greater than 70% sequence identitywith SEQ ID NO:2, preferably greater than 80%, more preferably greaterthan 90%, more preferably greater than 95% or, in another embodiment,has 98 to 100% sequence identity with SEQ ID NO:2. As described above,when describing the nucleotide in terms of SEQ ID NO: 1, the sequenceidentity may be slightly lower due to the degeneracy in the geneticcode. Also included within the definition of the target proteins areamino acid sequence variants of wild-type target proteins.

Portions of the HsKif12a nucleotide sequence may be used to identifypolymorphic variants, orthologs, alleles, and homologues of HsKif12a.This identification can be made in vitro, e.g., under stringenthybridization conditions and sequencing, or by using the sequenceinformation in a computer system for comparison with other nucleotidesequences. Sequence comparison can be performed using any of thesequence comparison algorithms discussed below, with PILEUP as apreferred algorithm.

As will be appreciated by those in the art, the target proteins can bemade in a variety of ways, including both synthesis de novo and byexpressing a nucleic acid encoding the protein.

Target proteins of the present invention may also be modified in a wayto form chimeric molecules comprising a fusion of a target protein witha tag polypeptide which provides an epitope to which an anti-tagantibody can selectively bind. The epitope tag is generally placed atthe amino or carboxyl terminus of the target protein. Provision of theepitope tag enables the target protein to be readily detected, as wellas readily purified by affinity purification. Various tag epitopes arewell known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 (see, Field et al. (1988) Mol. Cell. Biol.8:2159); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto (see, Evans et al., (1985) Molecular and CellularBiology, 5:3610); and the Herpes Simplex virus glycoprotein D (gD) tagand its antibody (see, Paborsky et al., (1990) Protein Engineering,3:547). Other tag polypeptides include the Flag-peptide (see, Hopp etal. (1988) BioTechnology 6:1204); the KT3 epitope peptide (see, Martineet al. (1992) Science, 255:192); tubulin epitope peptide (see, Skinner(1991) J. Biol. Chem. 266:15173); and the T7 gene 10 protein peptide tag(see, Lutz-Freyermuth et al. (1990) Proc. Natl. Acad. Sci. USA 87:6393.

The biological activity of any of the peptides provided herein can beroutinely confirmed by the assays provided herein such as those whichassay ATPase activity or microtubule binding activity. In oneembodiment, polymorphic variants, alleles, and orthologs, homologues ofHsKif12a are confirmed by using a ATPase or microtubule binding assaysas known in the art.

The isolation of biologically active HsKif12a for the first timeprovides a means for assaying for modulators of this kinesin superfamilyprotein. Biologically active HsKif12a is useful for identifyingmodulators of HsKif12a or fragments thereof and kinesin superfamilymembers using in vitro assays such as microtubule gliding assays, ATPaseassays (Kodama et al., J Biochem. 99:1465-1472 (1986); Stewart et al.,Proc. Nat'l Acad. Sci. USA 90:5209-5213 (1993)), and binding assaysincluding microtubule binding assays (Vale et al., Cell 42:39-50(1985)). In vivo assays and uses are provided herein as well. Alsoprovided herein are methods of identifying candidate agents which bindto HsKif12a and portions thereof.

As further described herein, a wide variety of assays, therapeutic anddiagnostic methods are provided herein which utilize the novel compoundsdescribed herein. The uses and methods provided herein, as furtherdescribed below have in vivo, in situ, and in vitro applications, andcan be used in medicinal, veterinary, agricultural and research basedapplications.

III. Isolation of the gene encoding HsKif12a

A. General Recombinant DNA Methods

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from mass spectroscopy, from sequenced proteins, fromderived amino acid sequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862(1981), using an automated synthesizer, as described in Van Devanter etal., Nucleic Acids Res. 12:6159-6168 (1984). Purification ofoligonculeotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson & Reanier, J Chrom.225:137-149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981).

B. Cloning Methods for the Isolation of Nucleotide Sequences EncodingHsKif12a

In general, the nucleic acid sequences encoding HsKif12a and relatednucleic acid sequence homologs are cloned from cDNA and genomic DNAlibraries or isolated using amplification techniques witholigonucleotide primers. Alternatively, expression libraries can be usedto clone HsKif12a and HsKif12a homologues by detected expressedhomologues immunologically with antisera or purified antibodies madeagainst HsKif12a that also recognize and selectively bind to theHsKif12a homologue. Finally, amplification techniques using primers canbe used to amplify and isolate HsKif12a from DNA or RNA. Amplificationtechniques using degenerate primers can also be used to amplify andisolate HsKif12a homologues. Amplification techniques using primers canalso be used to isolate a nucleic acid encoding HsKif12a. These primerscan be used, e.g., to amplify a probe of several hundred nucleotides,which is then used to screen a library for full-length HsKif12a.

Appropriate primers and probes for identifying the gene encodinghomologues of HsKif12a in other species are generated from comparisonsof the sequences provided herein. As described above, antibodies can beused to identify HsKif12a homologues. For example, antibodies made tothe motor domain of HsKif12a or to the whole protein are useful foridentifying HsKif12a homlogues.

To make a cDNA library, one should choose a source that is rich in themRNA of choice, e.g., HsKif12a. The mRNA is then made into cDNA usingreverse transcriptase, ligated into a recombinant vector, and introducedinto a recombinant host for propagation, screening and cloning. Methodsfor making and screening cDNA libraries are well known (see, e.g.,Gubler & Hoffman, Gene 25: 263-269); Sambrook et al., supra; Ausubel etal., supra).

For a genomic library, the DNA is extracted from the tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180-182 (1977). Colony hybridization is readout as generally described in Grunstein et al., Proc. Natl. Acad. Sci.USA, 72:3961-3965 (1975).

An alternative method of isolating HsKif12a nucleic acid and itshomologues combines the use of synthetic oligonucleotide primers andamplification of an RNA or DNA template (see U.S. Pat. Nos. 4,683,195and 4,683,202; PCR Protocols: A guide to Methods and Applications (Inniset al., eds. 1990)). Methods such as polymerase chain reaction andligase chain reaction can be used to amplify nucleic acid sequences ofHsKif12a directly from mRNA, from cDNA, from genomic libraries or cDNAlibraries. Degenerate oligonucleotides can be designed to amplifyHsKif12a homologues using the sequences provided herein. Restrictionendonuclease sites can be incorporated into the primers. Polymerasechain reaction or other in vitro amplification methods may also beuseful, for example, to clone nucleic acid sequences that code forproteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of HsKif12a encoding mRNA in physiologicalsamples, for nucleic sequencing or for other purposes. Genes amplifiedby the PCR reaction can be purified from agarose gels and cloned into anappropriate vector.

Gene expression of HsKif12a can also be analyzed by techniques known inthe art, e.g., reverse transcription and amplification of mRNA,isolation of total RNA or poly A+RNA, northern blotting, dot blotting,in situ hybridization, RNase protection, quantitative PCR, and the like.

Synthetic oligonucleotides can be used to construct recombinant HsKif12agenes for use as probes or for expression of protein. This method isperformed using a series of overlapping oligonucleotides usually 40-120bp in length, representing both the sense and nonsense strands of thegene. These DNA fragments are then annealed, ligated and cloned.Alternatively, amplification techniques can be used with precise primersto amplify a specific subsequence of the HsKif12a gene. The specificsubsequence is then ligated into an expression vector.

The gene for HsKif12a is typically cloned into intermediate vectorsbefore transformation into prokaryotic or eukaryotic cells forreplication and/or expression. The intermediate vectors are typicallyprokaryote vectors or shuttle vectors.

C. Expression in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene, such as those cDNAsencoding HsKif12a, it is important to construct an expression vectorthat contains a strong promoter to direct transcription, atranscription/translation terminator, and if for a nucleic acid encodinga protein, a ribosome binding site for translational initiation.Suitable bacterial promoters are well known in the art and described,e.g., in Sambrook et al. and Ausubel et al. Bacterial expression systemsfor expressing the HsKif12a protein are available in, e.g., E. coli,Bacillus sp., and Salmonella (Palva et al., Gene 22:229-235 (1983);Mosbach et al., Nature 302:543-545 (1983). Kits for such expressionsystems are commercially available. Eukaryotic expression systems formammalian cells, yeast, and insect cells are well known in the art andare also commercially available. The pET expression system (Novagen) isa preferred prokaryotic expression system.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is preferablypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the HsKif12a encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encodingHsKif12a and signals required for efficient polyadenylation of thetranscript, ribosome binding sites, and translation termination. Thenucleic acid sequence encoding HsKif12a may typically be linked to acleavable signal peptide sequence to promote secretion of the encodedprotein by the transformed cell. Such signal peptides would include,among others, the signal peptides from tissue plasminogen activator,insulin, and neuron growth factor, and juvenile hormone esterase ofHeliothis virescens. Additional elements of the cassette may includeenhancers and, if genomic DNA is used as the structural gene, intronswith functional splice donor and acceptor sites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc or histidine tags.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, cytomegalovirus vectors, papilloma virus vectors, and vectorsderived from Epstein Bar virus. Other exemplary eukaryotic vectorsinclude pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, andany other vector allowing expression of proteins under the direction ofthe SV40 early promoter, SV40 late promoter, CMV promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells. Some expression systemshave markers that provide gene amplification such as thymidine kinase,hygromycin B phosphotransferase, and dihydrofolate reductase.Alternatively, high yield expression systems not involving geneamplification are also suitable, such as using a baculovirus vector ininsect cells, with a HsKif12a encoding sequence under the direction ofthe polyhedrin promoter or other strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection or transformation methods are used to producebacterial, mammalian, yeast or insect cell lines that express largequantities of HsKif12a protein, which are then purified using standardtechniques (see, e.g., Colley et al., J Biol. Chem. 264:17619-17622(1989); Guide to Protein Purification, in Methods in Enzymology, vol.182 (Deutscher ed., 1990)).

Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.,132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology,101:347-362 (Wu et al., eds, 1983). Any of the well known procedures forintroducing foreign nucleotide sequences into host cells may be used.These include the use of calcium phosphate transfection, polybrene,protoplast fusion, electroporation, liposomes, microinjection, plasmavectors, viral vectors and any of the other well known methods forintroducing cloned genomic DNA, cDNA, synthetic DNA or other foreigngenetic material into a host cell (see, e.g., Sambrook et al., supra).It is only necessary that the particular genetic engineering procedureused be capable of successfully introducing at least one gene into thehost cell capable of expressing HsKif12a.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofHsKif12a, which is recovered from the culture using standard techniquesidentified below.

IV. Purification of HsKif12a Protein

Either naturally occurring or recombinant HsKif12a can be purified foruse in functional assays. In a preferred embodiment, the target proteinsare purified for use in the assays to provide substantially puresamples. Alternatively, the target protein need not be substantiallypure as long as the sample comprising the target protein issubstantially free of other components that can contribute to theproduction of ADP or phosphate.

The target proteins may be isolated or purified in a variety of waysknown to those skilled in the art depending on what other components arepresent in the sample. Standard purification methods includeelectrophoretic, molecular, immunological, and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, chromatofocussing, selectiveprecipitation with such substances as ammonium sulfate;and others (see,e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S.Pat. No. 4,673,641; Ausubel et al. supra; and Sambrook et al., supra).For example, the target protein can be purified using a standardanti-target antibody column. Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.A preferred method of purification is use of Ni-NTA agarose (Qiagen).

The expressed protein can be purified by standard chromatographicprocedures to yield a purified, biochemically active protein. Theactivity of any of the peptides provided herein can be routinelyconfirmed by the assays provided herein such as those which assay ATPaseactivity or microtubule binding activity. Biologically active targetprotein is useful for identifying modulators of target protein orfragments thereof and kinesin superfamily members using in vitro assayssuch as microtubule gliding assays, ATPase assays (Kodama et al., J.Biochem. 99:1465-1472 (1986); Stewart et al., Proc. Nat'l Acad. Sci. USA90:5209-5213 (1993)), and binding assays including microtubule bindingassays (Vale et al., Cell 42:39-50 (1985)), as described in detailbelow.

A. Purification of HsKif12a from Recombinant Bacteria

Recombinant proteins are expressed by transformed bacteria in largeamounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is a preferred method ofexpression. Bacteria are grown according to standard procedures in theart. Fresh or frozen bacteria cells are used for isolation of protein.Alternatively, it is possible to purify HsKif12a from bacteriaperiplasm. After HsKif12a is exported into the periplasm of thebacteria, the periplasmic fraction of the bacteria can be isolated bycold osmotic shock in addition to other methods known to skill in theart. To isolate recombinant proteins from the periplasm, the bacterialcells are centrifuged to form a pellet. The pellet is resuspended in abuffer containing 20% sucrose. To lyse the cells, the bacteria arecentrifuged and the pellet is resuspended in ice-cold 5 mM MgSO₄ andkept in an ice bath for approximately 10 minutes. The cell suspension iscentrifuged and the supernatant decanted and saved. The recombinantproteins present in the supernatant can be separated from the hostproteins by standard separation techniques well known to those of skillin the art.

Suitable purification schemes for some specific kinesins are outlined inU.S. Ser. No. 09/295,612, filed Apr. 20, 1999, hereby expresslyincorporated herein in its entirety for all purposes.

B. Standard Protein Separation Techniques For Purifying HsKif12aSolubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Differential Filtration

The molecular weight of HsKif12a can be used to isolated it fromproteins of greater and lesser size using ultrafiltration throughmembranes of different pore size (for example, Amicon or Milliporemembranes). As a first step, the protein mixture is ultrafilteredthrough a membrane with a pore size that has a lower molecular weightcut-off than the molecular weight of the protein of interest. Theretentate of the ultrafiltration is then ultrafiltered against amembrane with a molecular cut off greater than the molecular weight ofthe protein of interest. The recombinant protein will pass through themembrane into the filtrate. The filtrate can then be chromatographed asdescribed below.

Column Chromatography

HsKif12a can also be separated from other proteins on the basis of itssize, net surface charge, hydrophobicity, and affinity for ligands. Inaddition, antibodies raised against proteins can be conjugated to columnmatrices and the proteins immunopurified. All of these methods are wellknown in the art. It will be apparent to one of skill thatchromatographic techniques can be performed at any scale and usingequipment from many different manufacturers (e.g., Pharmacia Biotech).

V. Immunological Detection of HsKif12a

In addition to the detection of HsKif12a genes and gene expression usingnucleic acid hybridization technology, one can also use immunoassays todetect HsKif12a. Immunoassays can be used to qualitatively orquantitatively analyze HsKif12a. A general overview of the applicabletechnology can be found in Harlow & Lane, Antibodies: A LaboratoryManual (1988).

A. Antibodies to HsKif12a

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with HsKif12a are known to those of skill in the art (see,e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane,supra; Goding, Monoclonal Antibodies: Principles and Practice (2d ed.1986); and Kohler & Milstein, Nature 256:495-497 (1975). Such techniquesinclude antibody preparation by selection of antibodies from librariesof recombinant antibodies in phage or similar vectors, as well aspreparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989);Ward et al., Nature 341:544-546 (1989)).

A number of HsKif12a comprising immunogens may be used to produceantibodies specifically reactive with HsKif12a. For example, recombinantHsKif12a or a antigenic fragment thereof such as the motor domain, isisolated as described herein. Recombinant protein can be expressed ineukaryotic or prokaryotic cells as described above, and purified asgenerally described above. Recombinant protein is the preferredimmunogen for the production of monoclonal or polyclonal antibodies.Alternatively, a synthetic peptide derived from the sequences disclosedherein and conjugated to a carrier protein can be used an immunogen.Naturally occurring protein may also be used either in pure or impureform. The product is then injected into an animal capable of producingantibodies. Either monoclonal or polyclonal antibodies may be generated,for subsequent use in immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to HsKif12a. Whenappropriately high titers of antibody to the immunogen are obtained,blood is collected from the animal and antisera are prepared. Furtherfractionation of the antisera to enrich for antibodies reactive to theprotein can be done if desired (see Harlow & Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see Kohler & Milstein, Eur. J Immunol. 6:511-519 (1976)).Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods wellknown in the art. Colonies arising from single immortalized cells arescreened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse et al.,Science 246:1275-1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-HsKif12aproteins or even other homologous proteins from other organisms (e.g., Celegans unc-104 or human Kif1 A), using a competitive bindingimmunoassay. Specific polyclonal antisera and monoclonal antibodies willusually bind with a K_(d) of at least about 0.1 mM, more usually atleast about 1 μM, preferably at least about 0.1 μM or better, and mostpreferably, 0.01 μM or better.

Once HsKif12a specific antibodies are available, HsKif12a can bedetected by a variety of immunoassay methods. For a review ofimmunological and immunoassay procedures, see Basic and ClinicalImmunology (Stites & Terr eds., 7th ed. 1991). Moreover, theimmunoassays of the present invention can be performed in any of severalconfigurations, which are reviewed extensively in Enzyme Immunoassay(Maggio ed., 1980); and Harlow & Lane, supra.

B. Binding Assays

Antibodies can be used for treatment or to identify the presence ofHsKif12a having the sequence identity characteristics as describedherein. Additionally, antibodies can be used to identify modulators ofthe interaction between the antibody and HsKif12a as further describedbelow. While the following discussion is directed toward the use ofantibodies in the use of binding assays, it is understood that the samegeneral assay formats such as those described for “non-competitive” or“competitive” assays can be used with any compound which binds toHsKif12a such as microtubules or the compounds described in Ser. No.60/070,772.

In a preferred embodiment, HsKif12a is detected and/or quantified usingany of a number of well recognized immunological binding assays (see,e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168).For a review of the general immunoassays, see also Methods in CellBiology Volume 37: Antibodies in Cell Biology (Asai, ed. 1993); Basicand Clinical Immunology (Stites & Terr, eds., 7th ed. 1991).Immunological binding assays (or immunoassays) typically use an antibodythat specifically binds to a protein or antigen of choice (in this casethe HsKif12a or antigenic subsequence thereof). The antibody (e.g.,anti-HsKif12a) may be produced by any of a number of means well known tothose of skill in the art and as described above.

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled HsKif12a polypeptideor a labeled anti-HsKif12a antibody. Alternatively, the labeling agentmay be a third moiety, such a secondary antibody, that specificallybinds to the antibody/HsKif12a complex (a secondary antibody istypically specific to antibodies of the species from which the firstantibody is derived). Other proteins capable of specifically bindingimmunoglobulin constant regions, such as protein A or protein G may alsobe used as the label agent. These proteins exhibit a strongnon-immunogenic reactivity with immunoglobulin constant regions from avariety of species (see generally Kronval et al., J Immunol.111:1401-1406 (1973); Akerstrom et al., J Immunol. 135:2589-2542(1985)). The labeling agent can be modified with a detectable moiety,such as biotin, to which another molecule can specifically bind, such asstreptavidin. A variety of detectable moieties are well known to thoseskilled in the art.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,antigen, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 4° C. to 40° C.

Non-Competitive Assay Formats

Immunoassays for detecting HsKif12a in samples may be either competitiveor noncompetitive. Noncompetitive immunoassays are assays in which theamount of antigen is directly measured. In one preferred “sandwich”assay, for example, the anti-HsKif12a antibodies can be bound directlyto a solid substrate on which they are immobilized. These immobilizedantibodies then capture HsKif12a present in the test sample. HsKif12a isthus immobilized is then bound by a labeling agent, such as a secondHsKif12a antibody bearing a label. Alternatively, the second antibodymay lack a label, but it may, in turn, be bound by a labeled thirdantibody specific to antibodies of the species from which the secondantibody is derived. The second or third antibody is typically modifiedwith a detectable moiety, such as biotin, to which another moleculespecifically binds, e.g., streptavidin, to provide a detectable moiety.

Competitive assay formats

In competitive assays, the amount of HsKif12a present in the sample ismeasured indirectly by measuring the amount of a known, added(exogenous) HsKif12a displaced (competed away) from an anti-HsKif12aantibody by the unknown HsKif12a present in a sample. In one competitiveassay, a known amount of HsKif12a is added to a sample and the sample isthen contacted with an antibody that specifically binds to HsKif12a. Theamount of exogenous HsKif12a bound to the antibody is inverselyproportional to the concentration of HsKif12a present in the sample. Ina particularly preferred embodiment, the antibody is immobilized on asolid substrate. The amount of HsKif12a bound to the antibody may bedetermined either by measuring the amount of HsKif12a present in aHsKif12a/antibody complex, or alternatively by measuring the amount ofremaining uncomplexed protein. The amount of HsKif12a may be detected byproviding a labeled HsKif12a molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay the known HsKif12a, is immobilized on a solid substrate. Aknown amount of anti-HsKif12a antibody is added to the sample, and thesample is then contacted with the HsKif12a. The amount of anti-HsKif12aantibody bound to the known immobilized HsKif12a is inverselyproportional to the amount of HsKif12a present in the sample. Again, theamount of immobilized antibody may be detected by detecting either theimmobilized fraction of antibody or the fraction of the antibody thatremains in solution. Detection may be direct where the antibody islabeled or indirect by the subsequent addition of a labeled moiety thatspecifically binds to the antibody as described above.

Cross-reactivity determinations

Immunoassays in the competitive binding format can also be used forcrossreactivity determinations. For example, a protein at leastpartially encoded by SEQ ID NO:2 can be immobilized to a solid support.Proteins (e.g., C. elegans unc-104 or human Kif1A) are added to theassay that compete for binding of the antisera to the immobilizedantigen. The ability of the added proteins to compete for binding of theantisera to the immobilized protein is compared to the ability ofHsKif12a encoded by SEQ ID NO:2 to compete with itself. The percentcrossreactivity for the above proteins is calculated, using standardcalculations. Those antisera with less than 10% crossreactivity witheach of the added proteins listed above are selected and pooled. Thecross-reacting antibodies are optionally removed from the pooledantisera by immunoabsorption with the added considered proteins, e.g.,distantly related homologues.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps the protein of this invention, to the immunogenprotein (i.e., HsKif12a of SEQ ID NO:2). In order to make thiscomparison, the two proteins are each assayed at a wide range ofconcentrations and the amount of each protein required to inhibit 50% ofthe binding of the antisera to the immobilized protein is determined. Ifthe amount of the second protein required to inhibit 50% of binding isless than 10 times the amount of the protein encoded by SEQ ID NO:2 thatis required to inhibit 50% of binding, then the second protein is saidto specifically bind to the polyclonal antibodies generated to aHsKif12a immunogen.

Other assay formats

Western blot (immunoblot) analysis is used to detect and quantify thepresence of HsKif12a in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind HsKif12a. The anti-HsKif12a antibodiesspecifically bind to the HsKif12a on the solid support. These antibodiesmay be directly labeled or alternatively may be subsequently detectedusing labeled antibodies (e.g., labeled sheep anti-mouse antibodies)that specifically bind to the anti-HsKif12a antibodies.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:34-41 (1986)).

Reduction of non-specific binding

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²p), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), colorimetric labels such as colloidalgold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.) or other labels that can be detected by massspectroscopy, NMR spectroscopy, or other analytical means known in theart.

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecules (e.g., streptavidin)molecule, which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize HsKif12a, orsecondary antibodies that recognize anti-HsKif12a.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems which may be used, see U.S. Pat.No. 4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

VI. Assays for Modulators of the Target Protein

A. Functional Assays

Assays that can be used to test for modulators of the target proteininclude a variety of in vitro or in vivo assays, e.g., microtubulegliding assays, binding assays such as microtubule binding assays,microtubule depolymerization assays, and ATPase assays (Kodama et al.,J. Biochem. 99: 1465-1472 (1986); Stewart et al., Proc. Nat'l Acad. Sci.USA 90: 5209-5213 (1993); (Lombillo et al., J. Cell Biol. 128:107-115(1995); (Vale et al., Cell 42:39-50 (1985)).

Modulation is tested by screening for candidate agents capable ofmodulating the activity of the target protein comprising the steps ofcombining a candidate agent with the target protein, as above, anddetermining an alteration in the biological activity of the targetprotein. Thus, in this embodiment, the candidate agent should both bindto the target protein (although this may not be necessary), and alterits biological or biochemical activity as defined herein. The methodsinclude both in vitro screening methods and in vivo screening of cellsfor alterations in cell cycle distribution, cell viability, or for thepresence, morphology, activity, distribution, or amount of mitoticspindles, as are generally outlined above.

In a preferred embodiment, molecular motor activity is measured by themethods disclosed in Ser. No. 09/314,464, filed May 18, 1999, entitled“Compositions and assay utilizing ADP or phosphate for detecting proteinmodulators”, which is incorporated herein by reference in its entirety.More specifically, this assay detects modulators of any aspect of akinesin motor function ranging from interaction with microtubules tohydrolysis of ATP. ADP or phosphate is used as the readout for proteinactivity.

There are a number of enzymatic assays known in the art which use ADP asa substrate. For example, kinase reactions such as pyruvate kinases areknown. See, Nature 78:632 (1956) and Mol. Pharmacol. 6:31 (1970). Thisis a preferred method in that it allows the regeneration of ATP. In oneembodiment, the level of activity of the enzymatic reaction isdetermined directly. In a preferred embodiment, the level of activity ofthe enzymatic reaction which uses ADP as a substrate is measuredindirectly by being coupled to another reaction. For example, in oneembodiment, the method further comprises a lactate dehydrogenasereaction under conditions which normally allow the oxidation of NADH,wherein said lactate dehydrogenase reaction is dependent on the pyruvatekinase reaction. Measurement of enzymatic reactions by coupling is knownin the art. Furthermore, there are a number of reactions which utilizephosphate. Examples of such reactions include a purine nucleosidephosphorylase reaction. This reaction can be measured directly orindirectly. A particularly preferred embodiments utilizes the pyruvatekinase/lactate dehydrogenase system.

In one embodiment, the detection of the ADP or phosphate proceedsnon-enzymatically, for example, by binding or reacting the ADP orphosphate with a detectable compound. For example, phosphomolybdatebased assays may be used which involve conversion of free phosphate to aphosphomolybdate complex. One method of quantifying the phosphomolybdateis with malachite green. Alternatively, a fluorescently labeled form ofa phosphate binding protein, such as the E. coli phosphate bindingprotein, can be used to measure phosphate by a shift in itsfluorescence.

In addition, target protein activity can be examined by determiningmodulation of target protein in vitro using cultured cells. The cellsare treated with a candidate agent and the effect of such agent on thecells is then determined either directly or by examining relevantsurrogate markers. For example, characteristics such as mitotic spindlemorphology and cell cycle distribution can be used to determine theeffect.

Thus, in a preferred embodiment, the methods comprise combining a targetprotein and a candidate agent, and determining the effect of thecandidate agent on the target protein. Generally a plurality of assaymixtures are run in parallel with different agent concentrations toobtain a differential response to the various concentrations. Typically,one of these concentrations serves as a negative control, i.e., at zeroconcentration or below the level of detection.

As will be appreciated by those in the art, the components may be addedin buffers and reagents to assay target protein activity and giveoptimal signals. Since the methods allow kinetic measurements, theincubation periods can be optimized to give adequate detection signalsover the background.

In a preferred embodiment, an antifoam or a surfactant is included inthe assay mixture. Suitable antifoams include, but are not limited to,antifoam 289 (Sigma). Suitable surfactants include, but are not limitedto, Tween, Tritons, including Triton X-100, saponins, andpolyoxyethylene ethers. Generally, the antifoams, detergents, orsurfactants are added at a range from about 0.01 ppm to about 10 ppm.

A preferred assay design is also provided. In one aspect, the inventionprovides a multi-time-point (kinetic) assay, with at least two datapoints being preferred. In the case of multiple measurements, theabsolute rate of the protein activity can be determined.

B. Binding Assays

In a preferred embodiment, the binding of the candidate agent isdetermined through the use of competitive binding assays. In thisembodiment, the competitor is a binding moiety known to bind to thetarget protein, such as an antibody, peptide, binding partner, ligand,etc. Under certain circumstances, there may be competitive binding asbetween the candidate agent and the binding moiety, with the bindingmoiety displacing the candidate agent.

Competitive screening assays may be done by combining the target proteinand a drug candidate in a first sample. A second sample comprises acandidate agent, the target protein and a compound that is known tomodulate the target protein. This may be performed in either thepresence or absence of microtubules. The binding of the candidate agentis determined for both samples, and a change, or difference in bindingbetween the two samples indicates the presence of an agent capable ofbinding to the target protein and potentially modulating its activity.That is, if the binding of the candidate agent is different in thesecond sample relative to the first sample, the candidate agent iscapable of binding to the target protein.

In one embodiment, the candidate agent is labeled. Either the candidateagent, or the competitor, or both, is added first to the target proteinfor a time sufficient to allow binding. Incubations may be performed atany temperature which facilitates optimal activity, typically between 4and 40° C. Incubation periods are selected for optimum activity, but mayalso be optimized to facilitate rapid high throughput screening.Typically between 0.1 and 1 hour will be sufficient. Excess reagent isgenerally removed or washed away. The second component is then added,and the presence or absence of the labeled component is followed, toindicate binding.

In a preferred embodiment, the competitor is added first, followed bythe candidate agent. Displacement of the competitor is an indication thecandidate agent is binding to the target protein and thus is capable ofbinding to, and potentially modulating, the activity of the targetprotein. In this embodiment, either component can be labeled. Thus, forexample, if the competitor is labeled, the presence of label in the washsolution indicates displacement by the agent. Alternatively, if thecandidate agent is labeled, the presence of the label on the supportindicates displacement.

In an alternative embodiment, the candidate agent is added first, withincubation and washing, followed by the competitor. The absence ofbinding by the competitor may indicate the candidate agent is bound tothe target protein with a higher affinity. Thus, if the candidate agentis labeled, the presence of the label on the support, coupled with alack of competitor binding, may indicate the candidate agent is capableof binding to the target protein.

C. Candidate Agents

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof. Particularly preferred are peptides.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. In a preferred embodiment,the candidate agents are organic chemical moieties, a wide variety ofwhich are available in the literature.

D. Other Assay Components

The assays provided utilize target protein as defined herein. In oneembodiment, portions of target protein are utilized; in a preferredembodiment, portions having target protein activity as described hereinare used. In addition, the assays described herein may utilize eitherisolated target proteins or cells or animal models comprising the targetproteins.

A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc. which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Also,reagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding.

VII. Applications

The methods of the invention are used to identify compounds useful inthe treatment of cellular proliferation diseases. Disease states whichcan be treated by the methods and compositions provided herein include,but are not limited to, cancer (further discussed below), autoimmunedisease, arthritis, graft rejection, inflammatory bowel disease,proliferation induced after medical procedures, including, but notlimited to, surgery, angioplasty, and the like. It is appreciated thatin some cases the cells may not be in a hyper or hypo proliferationstate (abnormal state) and still require treatment. For example, duringwound healing, the cells may be proliferating “normally”, butproliferation enhancement may be desired. Similarly, as discussed above,in the agriculture arena, cells may be in a “normal” state, butproliferation modulation may be desired to enhance a crop by directlyenhancing growth of a crop, or by inhibiting the growth of a plant ororganism which adversely affects the crop. Thus, in one embodiment, theinvention herein includes application to cells or individuals afflictedor impending affliction with any one of these disorders or states.

The compositions and methods provided herein are particularly deemeduseful for the treatment of cancer including solid tumors such as skin,breast, brain, cervical carcinomas, testicular carcinomas, etc. Moreparticularly, cancers that may be treated by the compositions andmethods of the invention include, but are not limited to: Cardiac:sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma),myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogeniccarcinoma (squamous cell, undifferentiated small cell, undifferentiatedlarge cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchialadenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinorna,hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone:osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumorchordoma, osteochronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors; Nervous system: skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meninges (meningioma,meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform,oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological:uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumorcervical dysplasia), ovaries (ovarian carcinoma [serouscystadenocarcinorna, mucinous cystadenocarcinoma, unclassifiedcarcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma(embryonal rhabdomyosarcoma], fallopian tubes (carcinoma); Hematologic:blood (myeloid leukemia [acute and chronic], acute lymphoblasticleukemia, chronic lymphocytic leukemia, myeloproliferative diseases,multiple myeloma, myelodysplastic syndrome), Hodgkin's disease,non-Hodgkin's lymphoma [malignant lymphoma]; Skin: malignant melanoma,basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, molesdysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis;and Adrenal glands: neuroblastoma. Thus, the term “cancerous cell” asprovided herein, includes a cell afflicted by any one of the aboveidentified conditions.

Accordingly, the compositions of the invention are administered tocells. By “administered” herein is meant administration of atherapeutically effective dose of the candidate agents of the inventionto a cell either in cell culture or in a patient. By “therapeuticallyeffective dose” herein is meant a dose that produces the effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques. As is known in the art, adjustments for systemicversus localized delivery, age, body weight, general health, sex, diet,time of administration, drug interaction and the severity of thecondition may be necessary, and will be ascertainable with routineexperimentation by those skilled in the art. By “cells” herein is meantalmost any cell in which mitosis or meiosis can be altered.

A “patient” for the purposes of the present invention includes bothhumans and other animals, particularly mammals, and other organisms.Thus the methods are applicable to both human therapy and veterinaryapplications. In the preferred embodiment the patient is a mammal, andin the most preferred embodiment the patient is human.

Candidate agents having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a patient, asdescribed herein. Depending upon the manner of introduction, thecompounds may be formulated in a variety of ways as discussed below. Theconcentration of therapeutically active compound in the formulation mayvary from about 0.1-100 wt.%. The agents maybe administered alone or incombination with other treatments, i.e., radiation, or otherchemotherapeutic agents.

In a preferred embodiment, the pharmaceutical compositions are in awater soluble form, such as pharmaceutically acceptable salts, which ismeant to include both acid and base addition salts.

The pharmaceutical compositions can be prepared in various forms, suchas granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers; fillers suchas microcrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavoring agents; coloring agents; andpolyethylene glycol. Additives are well known in the art, and are usedin a variety of formulations. The administration of the candidate agentsof the present invention can be done in a variety of ways as discussedabove, including, but not limited to, orally, subcutaneously,intravenously, intranasally, transdermally, intraperitoneally,intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.In some instances, for example, in the treatment of wounds andinflammation, the candidate agents may be directly applied as a solutionor spray.

One of skill in the art will readily appreciate that the methodsdescribed herein also can be used for diagnostic applications. Adiagnostic as used herein is a compound or method that assists in theidentification and characterization of a health or disease state inhumans or other animals. More specifically, antibodies whichspecifically bind HsKif12a may be used for the diagnosis of disorderscharacterized by expression of HsKif12a or in assays to monitor patientsbeing treated with HsKif12a, or agonists, antagonists, or inhibitors ofHsKif12a. Diagnostic assays include methods which utilize the antibodyand a label to detect HsKif12a in human body fluids or in extracts ofcells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by covalent or non-covalent attachmentof a reporter molecules. A wide variety of reporter molecules are knownin the art and may be used.

A variety of protocols for measuring HsKif12a, including ELISAs, RIAs,and FACS, are known in the art and provide a basis for diagnosingaltered or abnormal levels of HsKif12a expression. Normal or standardvalues for HsKif12a expression are established by coming body fluids orcell extracts taken from normal mammalian subjects, preferably human,with antibody to HsKif12a under conditions suitable for complexformation. The amount of standard complex formation may be quantitatedby various methods, preferably by photometric means. Quantities ofHsKif12a expressed in subject, control, and disease samples frombiopsied tissues can be compared with the standard values. Deviationbetween standard and subject values establishes the parameters fordiagnosing disease.

In another embodiment of the invention, the polynucleotides encodingHsKif12a may be used for diagnostic purposes. The polynucleotides whichmay be used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which HsKif12a may becorrelated with disease. The diagnostic assay may be used to determineabsence, presence, and excess expression of HsKif12a, and to monitorregulation of HsKif12a levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences encodingHsKif12a or closely related molecules may be used. The specificity ofthe probe, whether it's made from a highly specific region or from aless specific region, and the stringency of the hybridization oramplification (maximal, high, intermediate, or low) will determinewhether the probe identifies only naturally occurring sequences encodingHsKif12a, allelic variants, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably have at least 50% sequence identity to any of theHsKif12a encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequence of SEQID NO: 1 or from genomic sequences including promoters, enhancers, andintrons of the HsKif12a gene.

Means for producing specific hybridization probes for DNAs encodingHsKif12a include the cloning of polynucleotide sequences encodingHsKif12a or derivatives thereof into vectors for the production of mRNAprobes. Such vectors are known in the art, are commercially available,and may be used to synthesize RNA probes in vitro by means of theaddition of the appropriate RNA polymerases and the appropriate labelednucleotides. Hybridization probes may be labeled by a variety ofreporter groups, for example, by radionuclides such as ³²p or ³⁵S, or byenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems and the like.

In a particular aspect, the nucleotide sequences encoding HsKif12a maybe useful in assays that detect the presence of associated disorders.The nucleotide sequences encoding HsKif12a may be labeled by standardmethods and added to a fluid or tissue sample from a patient underconditions suitable for the formation of hybridization complexes. Aftera suitable incubation period, the sample is washed and the signal isquantitated and compared with a standard value. If the amount of signalis significantly altered in comparison to a control sample then thepresence of altered levels of nucleotide sequences encoding HsKif12a inthe sample indicates the presence of the associated disorder. Suchassays may also be used to evaluate the efficacy of a particulartherapeutic treatment regimen in animal studies, in clinical trials, orto monitor the treatment of an individual patient.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding HsKif12a may involve the use of PCR. These oligomersmay be chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding HsKif12a, or a fragment of a polynucleotide complementary tothe polynucleotide encoding HsKif12a, and will be employed underoptimized conditions for identification of a specific gene or condition.Oligomers may also be employed under less stringent conditions fordetection or quantitation of closely related DNA or RNA sequences.

Methods which may be used to quantitate the expression of HsKif12ainclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves.The speed of quantitation of multiple samples may be accelerated byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometer or colorimetricresponse gives rapid quantitation.

One of skill in the art will readily appreciate that the methodsdescribed herein also can be used for diagnostic applications. Adiagnostic as used herein is a compound or method that assists in theidentification and characterization of a health or disease state inhumans or other animals.

The present invention also provides for kits for screening formodulators of the target protein. Such kits can be prepared from readilyavailable materials and reagents. For example, such kits can compriseany one or more of the following materials: biologically active targetprotein, reaction tubes, and instructions for testing activity of thetarget protein. Preferably, the kit contains biologically active targetprotein. A wide variety of kits and components can be prepared accordingto the present invention, depending upon the intended user of the kitand the particular needs of the user. For example, the kit can betailored for ATPase assays, microtubule gliding assays, or microtubulebinding assays.

VIII. Examples

This assay is based on detection of ADP production from a targetprotein's microtubule stimulated ATPase. ATP production is monitored bya coupled enzyme system consisting of pyruvate kinase and lactatedehydrogenase. Under the assay conditions described below, pyruvatekinase catalyzes the conversion of ADP and phosphoenol pyruvate topyruvate and ATP. Lactate dehydrogenase then catalyzes theoxidation-reduction reaction of pyruvate and NADH to lactate and NAD+.Thus, for each molecule of ADP produced, one molecule of NADH isconsumed. The amount of NADH in the assay solution is monitored bymeasuring light absorbance at a wavelength of 340 nm.

The final 25 μl assay solution consists of the following: 5 μg/ml targetprotein, 30 μg/ml microtubules, 5 μM Taxol, 0.8 mM NADH, 1.5 mMphosphoenol pyruvate, 3.5 U/ml pyruvate kinase, 5 U/ml lactatedehydrogenase, 25 mM Pipes/KOH pH 6.8, 2 mM MgCl₂, 1 mM EGTA, 1 mM MDTT,0.1 mg/ml BSA, 0.001% antifoam 289, and 1 mM ATP.

Potential candidate agents are dissolved in DMSO at a concentration ofabout 1 mg/ml and 0.5 μof each chemical solution is dispensed into asingle well of a clear 384 well plate. Each of the 384 wells are thenfilled with 20 μl of a solution consisting of all of the assaycomponents described above except for ATP. The plate is agitated at ahigh frequency. To start the assay, 5 μl of a solution containing ATP isadded to each well. The plate is agitated and the absorbance is read at340 nm over various time intervals. The assay is run at roomtemperature.

The assay components and the performance of the assay are optimizedtogether to match the overall read time with the rate of the targetprotein's ADP production. The read time should be long enough for therate of NADH consumption to reach steady state beyond an initial lagtime of several seconds.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety.

2 1 823 DNA Human 1 tttctcctgc actgttttca cctttggcca gacgggctctgggaagacct acaccctgac 60 tggaccccct ccccaggggg agggggtgcc tgtaccccccagcctggctg gcatcatgca 120 gaggaccttc gcctggctgt tggaccgcgt gcagcacctgggtgcccctg tcacccttcg 180 cgcctcttat ctggagatct acaatgagca ggttcgggacttgctgagcc tggggtctcc 240 ccggcccctc cctgttcgct ggaacaagac tcggggcttctatgtggagc agctgcgggt 300 ggtggaattt gggagtctgg aggccctgat ggaacttttgcaaacgggtc tcagccgtcg 360 aaggaactca gcccacaccc tgaaccaggc ctccagccgaagccatgccc tgctcaccct 420 ttacatcagc cgtcaaactg cccagcagat gccttctgtggaccctgggg agccccctgt 480 tggtgggaag ctgtgctttg tggacctggc aggcagtgagaaggtagcag ccacgggatc 540 ccgtggggag ctgatgcttg aggctaacag catcaaccgaagcctgctgg ccctgggtca 600 ctgcatctcc ctgctgctgg acccacagcg gaagcagagccacatccctt tccgggacag 660 caagctcacc aagttgctgg cagactcact gggagggcgcggggtcaccc tcatgaggag 720 cgctcagggg gagtgcactc cccaggtggc ctgcgtgtccccctcagccc agtgccttcc 780 tgagactctc agcaccctgc gatatgcaag ccgagctcagcgg 823 2 274 PRT Human 2 Phe Ser Cys Thr Val Phe Thr Phe Gly Gln ThrGly Ser Gly Lys Thr 1 5 10 15 Tyr Thr Leu Thr Gly Pro Pro Pro Gln GlyGlu Gly Val Pro Val Pro 20 25 30 Pro Ser Leu Ala Gly Ile Met Gln Arg ThrPhe Ala Trp Leu Leu Asp 35 40 45 Arg Val Gln His Leu Gly Ala Pro Val ThrLeu Arg Ala Ser Tyr Leu 50 55 60 Glu Ile Tyr Asn Glu Gln Val Arg Asp LeuLeu Ser Leu Gly Ser Pro 65 70 75 80 Arg Pro Leu Pro Val Arg Trp Asn LysThr Arg Gly Phe Tyr Val Glu 85 90 95 Gln Leu Arg Val Val Glu Phe Gly SerLeu Glu Ala Leu Met Glu Leu 100 105 110 Leu Gln Thr Gly Leu Ser Arg ArgArg Asn Ser Ala His Thr Leu Asn 115 120 125 Gln Ala Ser Ser Arg Ser HisAla Leu Leu Thr Leu Tyr Ile Ser Arg 130 135 140 Gln Thr Ala Gln Gln MetPro Ser Val Asp Pro Gly Glu Pro Pro Val 145 150 155 160 Gly Gly Lys LeuCys Phe Val Asp Leu Ala Gly Ser Glu Lys Val Ala 165 170 175 Ala Thr GlySer Arg Gly Glu Leu Met Leu Glu Ala Asn Ser Ile Asn 180 185 190 Arg SerLeu Leu Ala Leu Gly His Cys Ile Ser Leu Leu Leu Asp Pro 195 200 205 GlnArg Lys Gln Ser His Ile Pro Phe Arg Asp Ser Lys Leu Thr Lys 210 215 220Leu Leu Ala Asp Ser Leu Gly Gly Arg Gly Val Thr Leu Met Arg Ser 225 230235 240 Ala Gln Gly Glu Cys Thr Pro Gln Val Ala Cys Val Ser Pro Ser Ala245 250 255 Gln Cys Leu Pro Glu Thr Leu Ser Thr Leu Arg Tyr Ala Ser ArgAla 260 265 270 Gln Arg

What is claimed is:
 1. An isolated nucleic acid sequence encoding amicrotubule motor protein, wherein the motor protein has the followingproperties: (i) the protein's activity includes microtubule stimulatedATPase activity; and (ii) the protein has a sequence that has greaterthan 90% amino acid sequence identity to SEQ ID NO:2 as measured using asequence comparison algorithm.
 2. An isolated nucleic acid sequence ofclaim 1, wherein the protein specifically binds to polyclonal antibodiesto a protein comprising SEQ ID NO:2.
 3. An isolated nucleic acidsequence of claim 1, wherein the nucleic acid encodes SEQ ID NO:2.
 4. Anisolated nucleic acid sequence of claim 1, wherein the nucleic acid hasa nucleotide sequence of SEQ ID NO:1.
 5. An isolated nucleic acidsequence of claim 1, wherein the nucleic acid selectively hybridizesunder high stringent hybridization conditions to SEQ ID NO:1.
 6. Anexpression vector comprising a nucleic acid encoding a microtubule motorprotein, wherein the motor protein has the following properties: (i) theprotein's activity includes microtubule stimulated ATPase activity; and(ii) the protein has a sequence that has greater than 90% amino acidsequence identity to SEQ ID NO:2 as measured using a sequence comparisonalgorithm.
 7. A host cell transfected with the vector of claim 6.